Fusion protein containing glicetin-1 as well as preparation method and application

A technology of glucagon and fusion protein, which is applied in the field of fusion protein containing glucagon-like peptide-1 and its preparation, to achieve the effects of stimulating secretion, improving glucose tolerance, and protecting pancreatic beta cells

Active Publication Date: 2012-07-18
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the hypoglycemic test of the GLP-1 fusion polypeptide provided by this invention in the diabetic animal model db / db mice shows that there is still room for improvement in its hypoglycemic effect

Method used

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  • Fusion protein containing glicetin-1 as well as preparation method and application
  • Fusion protein containing glicetin-1 as well as preparation method and application
  • Fusion protein containing glicetin-1 as well as preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] A recombinant plasmid containing the sequence encoding the fusion protein of glucagon-like peptide-1 or its derivatives (abbreviated as GLP-1 fusion protein) was constructed.

[0046] (1) PCR and enzyme digestion of the nucleotide sequence encoding the GLP-1 fusion protein

[0047] According to the structural characteristics of the GLP-1 fusion protein, the codon preference of Escherichia coli was used to design primers, and the primers were commercially synthesized by Shanghai Bioengineering Co., Ltd.

[0048] The primer sequences are as follows:

[0049] Upstream Primer 1 (UP1): 5'-GCGC GAT CCG CTG CCG CAT AGCCAT CGC GCC CAT AGC CTG CCG CCG-3’

[0050] Upstream primer 2 (UP2): 5'-CAT AGC CTG CCG CCG TTC AAC CCG AAG ACGCCG CAC GCT GAA GGT ACC-3'

[0051] Downstream primer (DP): 5'-TCATCCGCCAAAACAGCCAAG-3';

[0052] Using the overlap extension PCR method, the plasmid pMFH-GLP-1(7-37) (this plasmid has been applied for an invention patent application with the applic...

Embodiment 2

[0072] Example 2: Fermentation expression of pMFH-GLP-1 fusion protein

[0073] The recombinant plasmid pMFH-GLP-1Der was extracted, and further transformed into Escherichia coli BL21(DE3) (Bao Biology (Dalian) Bioengineering Co., Ltd.) to obtain genetically engineered expression bacteria. Through shaking flask culture and gradually amplifying, culture and ferment GLP-1 fusion protein expressing bacteria in 5L fermenter. The medium used is 2YT medium, the formula is: tryptone 16g / L, lactose 10g / L, NaCl 5g / L; use NaOH to adjust the pH to 7.0, and sterilize in the fermenter. The sterilization conditions are: 121°C, 20min . The fermentation culture conditions are as follows: the inoculum size is 1% by volume; the culture temperature is 37° C.; the liquid filling volume is 80% by volume; the concentration of ampicillin (Amp) is 100 mg / L. Cultivate to the middle and late stages of logarithmic growth, add 0.6mM IPTG, induce time for 6h, and collect the bacteria by centrifugation a...

Embodiment 3

[0074] Example 3: Separation and purification to obtain GLP-1 fusion protein

[0075] (1) Collect the fermented cells by centrifugation and resuspend with lysate. The formula of lysate is: 50mmol / LNaH 2 PO 4 , 300mmol / L NaCL, 10mmol / L imidazole (imidazole), adjust the pH to 8.0 with NaOH. Ultrasonic disruptor (DF-6P3C Ultrasonic Cell Disruptor, Ningbo Xinzhike Research Institute) was used to disrupt the bacterial cells in an ice bath, turned on for 5 s, stopped for 5 s, and worked for 20 min. 10000rpm, 4°C, 30min, centrifuge to break up the bacteria, and collect the supernatant.

[0076] (2) Purify the supernatant collected in step (1) with a Ni-NTA Agarose (nickel-charged resin, QIAGEN) affinity column, wash away impurity proteins with a lysate containing 10mM imidazole, and then use a lysate containing 250mM imidazole The lysate (except for the different imidazole concentration, other components are the same as the above lysate) elutes the target protein.

[0077] (3) Hy...

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Abstract

The invention discloses a fusion protein containing glicetin-1 (GLP-1) as well as a preparation method and application. The fusion protein has the amino acid sequence shown in SEQ ID NO.1. According to the invention, an amino acid with known sequence is mutated by a gene engineering method to obtain the nucleotide sequence encoding the fusion protein provided by the invention, then the nucleotide sequence is cloned to an expression carrier, and expression and purification are carried out to obtain the fusion protein. By adopting the fusion protein containing GLP-1, the GLP-1 has better stability and duration in physiological action; furthermore, pharmacodynamic result shows that the fusion protein has significant effect of reducing blood sugar; and in addition, by long-term injection of the fusion protein, the sugar tolerance is significantly improved, the insulin secretion is stimulated, pancreatic island beta cells are protected and the development of the diabetes course is delayed. Therefore, the GLP-1 fusion protein has extraordinary advantages and potential to be applied to clinical treatment.

Description

technical field [0001] The invention relates to a fusion protein, in particular to a fusion protein containing glucagon-like peptide-1, its preparation method and application. Background technique [0002] Diabetes has become the third largest non-communicable disease after cardiovascular diseases and tumors. The World Health Organization (WHO) predicts that there will be more than 360 million diabetics in the world in 2030, of which type II diabetes accounts for more than 90%. Type II diabetes is non-insulin-dependent diabetes mellitus, which is a common disease with increasing incidence rate year by year. [0003] Glucagon-like peptide-1 (Glucagon-like peptide-1, GLP-1) analogues and their derivatives show broad prospects in the treatment of type II diabetes. In the L cells of the small intestinal mucosa, proglucagon is enzymatically hydrolyzed to produce non-biologically active GLP-1(1-37), and GLP-1(1-37) is further hydrolyzed to remove 6 amino acids at the N-terminal t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/26A61K47/48A61P3/10A61P3/04A61P3/00C12R1/19
Inventor 李弘剑王从峰冉艳红苏正定周天鸿
Owner JINAN UNIVERSITY
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